Location: Characterization and Interventions for Foodborne Pathogens
2024 Annual Report
Objectives
Objective 1: Development and validation of sample preparation methods for the detection of foodborne bacterial pathogens and toxins.
Subobjective 1A: Generate, evaluate, and transfer a new class of magnetic materials for the effective partitioning and concentration of bacteria from large volume samples.
Subobjective 1B: Adaptation of surface chemistry for the effective separation and concentration of pathogens from foods.
Objective 2: Development and validation of rapid screening methods for foodborne bacterial pathogens and toxins, and identification of biomarkers.
Subobjective 2A: Transfer methods to quantify foodborne pathogens.
Subobjective 2B: Application of droplet digital PCR (ddPCR) to pathogen detection and quantitation.
Subobjective 2C: Improve and expand the utility to aid in the transfer of the immunoelectrochemical biosensor technology for the detection of toxins and pathogens in food.
Objective 3: Rapid identification, genotyping, and sequence analysis of foodborne bacterial pathogens.
Subobjective 3A: Generate, evaluate, and transfer a novel AlphaLISA to confirm the presence of select foodborne pathogens.
Subobjective 3B: Generate pathogen databases and improve the accuracy of the BEAM (formerly BActerial Rapid Detection using Optical scattering Technology or BARDOT) system.
Subobjective 3C: Rapid identification and enumeration of both E. coli O157:H7 and Salmonella by MPN combined with multiplex qPCR.
Subobjective 3D: Rapid identification of Campylobacter and Salmonella by target amplification and next generation sequencing using portable MinION sequencer.
Subobjective 3E: Whole genome sequencing analysis of the phylogenesis, virulence factors and antimicrobial resistance of Campylobacter spp. from meat samples.
Approach
The primary goal of this plan is to develop rapid screening and identification methods for top, foodborne bacterial pathogens (Shiga toxin producing E. coli or STEC, Salmonella serotypes, L. monocytogenes, etc.). Testing for specific pathogens in select foods is sometimes an intermittent demand as gaps in methodology and needs may arise. However, the technology to be generated in this plan will proactively be suited for quick adaption to these needs typically only requiring, for example, substitution of a recognition element (e.g., antibody or DNA primer) or bioinformatics-based mining for unique stretches of DNA sequences.
The detection of low levels of pathogens is complicated due to a gap in screening platform sensitivity, therefore we will increase sample volumes in order to elevate the amount of pathogens per test, especially when culture enrichment is not suitable (e.g., for rapid, field-based testing for very low concentrations of bacterial adulterants). To achieve this, novel sample preparation techniques will be key for rapid concentration of bacteria typically from aqueous homogenates. Subsequently, higher levels of detection sensitivity are expected as well as quantitation of extremely low levels (~1 cell/100 mL) of pathogens as needed for real-time testing. Assay times should be a few minutes to = 2 hours. Also, enhanced detection systems will be needed to bypass growth enrichment and achieve the desired detection levels. Furthermore, numerous biomarkers and the potential for false positive results using cross-reacting biorecognition elements (such as antibodies) will require multiplex detection techniques. However, for food contaminated with very low levels of target pathogens, detection may benefit from enrichment for accuracy thus avoiding false negative results. Therefore, conditions warranting brief enrichment prior to detection will be addressed.
Methods will initially be developed with culture media or buffer as the sample matrix, and then extended to application with food (primarily ground meats). Assay performance of developed methods will be compared against “gold standard” methods initially with reliance on bacterial enumeration. Evntually, developed methods will be tested using FSIS samples in comparison to state-of-the-art methods. Yet the 5-year time frame for this plan may not allow for full scale, multi-laboratory validation of methods. Hence optimization of robust and reproducible technologies may better merit the time and financial investment associated with such validation. Eventually, testing will move to the field first off-line, then in-line (for some methods) in regulated environments. It is expected that multitudes of tests will be conducted given that most samples are negative for contamination by pathogens. Regulatory and perhaps legal guidance will be anticipated to be critical since validation testing will lead to remediation or recall if zero-tolerance organisms are detected or if certain instances of positive samples are discovered.
Progress Report
Experiments were completed for a peer-reviewed manuscript describing a cost-effective method for the isolation of pathogens of interest from foods using the ARS developed magnetic capture device (USDA filed patent application 62/737,212). This manuscript will include the incorporation of a photocleavable linkage on the magnetic capture device as release of captured biological material via avidin-biotin binding using a competitive analog for biotin, desthiobiotin, was demonstrated to not be successful.
Two-dimensional nanomaterials, MXenes, were prepared into coatings on food contact surfaces and assessed for inhibition of bacterial attachment and antimicrobial activity. To functionally process the MXenes in a manner that did not require the addition of chemical additives that are not approved for food contact, ARS led the development of a novel dispersion process. The processed nanomaterials were then capable of being processed into coatings which utilized their morphologies to control the topography of the surfaces. The results were included in a peer-reviewed manuscript. Therefore MXenes show promise beyond assisting foodborne pathogen detection efforts.
The commercially available oCelloScope is a unique, high throughput live-cell imaging system for sensitive and detailed monitoring of biological growth and development. The oCelloScope automatically acquires and analyzes cell images in standard microtiter plates, however the device lacks the ability to quantify the concentration of bacteria in a specific volume. Research was conducted to develop a method which can use the images captured to quantify the concentration of bacteria in the sample within a range of 10e3 to 10e7 CFU/mL. The accuracy was benchmarked against a conventional microbial drop plate method as well as against droplet digital PCR. The experiments have been completed and a peer-reviewed manuscript will be submitted before the close of the fiscal year.
Research conducted using BioRad’s QX600 droplet digital PCR (ddPCR) instrument demonstrated the ability to detect Salmonella from a poultry matrix. This technology, when paired with a modified version of Pathotrak’s sample preparation system, was revealed to detect 1 CFU/ g of Salmonella in chicken samples without the need for enrichment. Testing was performed using 325-gram samples of chicken meat, with initial studies demonstrating an ability to differentiate 1 CFU/ g from 10 CFU/ g of chicken. The results of these studies will be presented at the AOAC meeting in Baltimore in August, with members of the FSIS Microbiology Issues Steering Group in attendance.
ARS led the method for delaminating multilayer MXene (ML-Mxene) using high pressure homogenization (HPH) and the prepared HPH-delaminated Mxene (HPH-Mxene) into electrodes for electrochemical biosensing. The current stage of development is proof-of-concept, having demonstrated the successful delamination of Ti3C2Tx multilayer powder into stable aqueous suspensions of few and single layer flakes through appropriate material characterization. Pristine HPH-Mxene electrodes were fabricated and have demonstrated the potential to be used as electrochemical sensors for the quantification of oxidized-3,3’,5,5’-tetramethylbenzdine (TMB), an enzymatic substrate used in an indirect immunoelectrochemical flow-through detection method (ARS patent pending) for foodborne pathogens with improved performance. Preliminary results using Square Wave Voltammetry showed an increase in current response (100X) from oxidized-TMB using HPH-MXene electrodes compared to a standard glassy carbon (GC) electrode. Further, electrochemical characterization indicated HPH-MXene to have good performance as an energy storage material. The results led to a patent application submitted in collaboration between ARS and the collaborating institution.
Research was completed for the development of a loop-mediated isothermal amplification (also known as LAMP) method to quantify Salmonella in contaminated, raw, and frozen breaded chicken products at low (0.1-1.0 CFU/g) levels. Additional data is being collected and will be incorporated into a peer-reviewed manuscript to fully meet the 48-month milestone.
A bioinformatics pipeline was developed to rapidly identify virulence genes of interest in samples sequenced with the MinION, long read DNA sequencing platform. The method was applied to ground beef and E. coli and Salmonella contamination were demonstrated to be detected.
A peer reviewed manuscript on the functional analysis of antimicrobial resistance and virulence was published. In addition to this, a peer-reviewed manuscript that detailed the process utilized to isolate the microorganisms from retail meat products was published to help transfer the methodology to the global scientific community.
Accomplishments
1. Nanomaterial derived surfaces inhibit bacterial adhesion in food processing. A collaboration led by ARS researchers in Wyndmoor, Pennsylvania, has yielded a revolutionary functional coating, leveraging two-dimensional nanoparticles, to tackle the critical challenge of bacterial adhesion in food processing environments, a persistent threat leading to foodborne illness outbreaks. This innovation builds upon ARS's patent-pending, environmentally friendly nanomaterial processing technique, guaranteeing the resulting coating is composed solely of Generally Recognized As Safe (GRAS) materials. This ensures exceptional food contact safety while achieving a minimum 3-log reduction in key pathogens like Listeria, E. coli, and Salmonella on commonly encountered food contact surfaces. This technology presents an advancement in targeted pathogen mitigation through novel material interactions and surface engineering. It empowers food processors with safer environments, potentially reducing recalls and safeguarding consumers, while granting regulators a powerful tool for enhanced hygiene and public health protection.
2. Expansion of the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA). Enzyme-linked immunosorbent assays (ELISAs) are one of the most widely adopted detection platforms for quantifying analytes in biological samples and are known for their simplicity and sensitivity. However, their typical dynamic range is relatively narrow and their automation is hampered by mandatory wash steps. The “no wash” alternative, known as the AlphaLISA, overcomes these technical hurdles. ARS researchers at Wyndmoor, Pennsylvania, have created an innovative platform, known as the oligo-Alpha, that utilized the same principles as the AlphaLISA yet is capable of detecting nucleic acids instead of proteins. This technology has the advantage of a large dynamic range, rapid testing time, reduced hands-on workflow resulting from the ability to sequentially overlay the reagents, and is readily automated. Despite the recency of its development, the oligo-Alpha has readily been adapted to the detection of Listeria monocytogenes and is currently being applied to other foodborne pathogens within the Campylobacter genus.
Review Publications
He, Y., Kanrar, S., Reed, S., Lee, J., Capobianco Jr, J.A. 2024. Whole genome sequences, de novo assembly, and annotation of antibiotic resistant Campylobacter jejuni strains S27, S33, and S36 newly isolated from chicken meat. Microorganisms. 12(1):159. https://doi.org/10.3390/microorganisms12010159.
Counihan, K.L., Kanrar, S., Tilman, S.M., Capobianco Jr, J.A., Armstrong, C.M., Gehring, A.G. 2024. Detection of Escherichia coli O157:H7 in ground beef using long-read sequencing. Foods. 13(6):828. https://doi.org/10.3390/foods13060828.
Inman, C.A., Shevchuk, K., Anayee, M., Hammill, B., Lee, J., Saraf, M., Shuck, C., Armstrong, C.M., He, Y., Jin, Z.T., Shekhirev, M., Capobianco Jr, J.A., Gogotsi1, Y. 2023. High-yield and high-throuhput delamination of multilayer MXene via high-pressure homogenization. Chemical Engineering Journal. 475:146089. https://doi.org/10.1016/j.cej.2023.146089.
Counihan, K.L., Kanrar, S., Tilman, S.M., Gehring, A.G. 2023. Evaluation of long-read sequencing simulators to assess real-world applications for food safety. Foods. 13(1):16. https://doi.org/10.3390/foods13010016.
Armstrong, C.M., Capobianco Jr, J.A., Lee, J. 2024. Magnetic capture device for large volume sample analysis. PLOS ONE. https://doi.org/10.1371/journal.pone.0297806.
Armstrong, C.M., Capobianco Jr, J.A., Nguyen, S.C., Guragain, M., Liu, Y. 2024. High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA. Scientific Reports. 14:7026. https://doi.org/10.1038/s41598-024-56911-8.